Endothelial nitric oxide synthase alterations are independent of turbulence in the aorta of patients with a unicuspid aortic valve

Abstract

Objectives: Certain aortic valve malformations predispose to ascending aortic aneurysm, although the mechanisms are incompletely understood. The aim of this study was to determine whether turbulence across the unicuspid aortic valve (UAV) contributes to regional differences in endothelial nitric oxide (eNOS) signaling in the ascending aortic wall.

Methods: Samples were collected intraoperatively from the convex and concave ascending aortic wall from 64 patients with tricuspid aortic valves (TAVs; 25 nondilated, 17 dilated), or UAVs (9 nondilated, 13 dilated).

Results: In normal-sized aortas, eNOS protein was decreased in UAV compared with TAV (P = .02) whereas mRNA was similar (P = .62). eNOS protein was increased in UAV-dilated aortas compared with UAV-nondilated aortas (P = .04), whereas dilatation had no impact on eNOS protein levels in TAV aortas (P = .73). Comparing only aneurysmal aortas, we found no difference in eNOS mRNA or protein between dilated TAV and UAV aortas (P = .26, P = .76). For eNOS mRNA and protein levels in normal and dilated UAV-associated aortas, no differences were found between concavity and convexity (all P > .05). This differed from dilated TAV aortas, which showed decreased eNOS mRNA in the convexity (P = .004) whereas eNOS protein levels were similar (P = .75).

Conclusions: eNOS downregulation is observed in the UAV-associated ascending aorta and is apparently independent of dilatation. No regional differences were found, however, which would be expected if eNOS changes occur due to wall shear stress. This implies a congenital defect in eNOS signaling that may be stronger than turbulence-induced expression patterns. Further research should define the role of eNOS in aortopathy associated with aortic valve disease.

Keywords: AV, aortic valve; BAV, bicuspid aortic valve; NOS3, nitric oxide synthase 3; PCR, polymerase chain reaction; TAV, tricuspid aortic valve; UAV, unicuspid aortic valve; aortic valve disease; aortopathy; ascending aorta; eNOS; eNOS, endothelial nitric oxide synthase; mRNA, messenger RNA; unicuspid aortic valve.

Graphical abstract

Regional assessment of UAV-associated aortas 13 suggests turbulence-independent eNOS changes.

Figure 1

Endothelial nitric oxide synthase (eNOS) protein levels are downregulated in the UAV-associated nondilated ascending aorta, whereas phosphorylated eNOS is increased in the media layer. A, Box and whisker plot depicting the mRNA expression of NOS3 (eNOS) in TAV- and UAV-associated ascending aortic tissue from normal (nondilated) ascending aortas (P = .62). B, Box and whisker plot and representative Western blot images depicting the decreased protein concentration of eNOS in UAV-associated ascending aortic tissue compared with TAV from normal (nondilated) ascending aortas (P = .02). C, Box and whisker plots depicting total eNOS protein levels in the intima (P = .04), media (P = .03), and adventitia (P = .81) from nondilated TAV and UAV aortas. D, Top: Fluorescent images of ascending aortic tissue stained for phosphorylated-eNOS (red) and counterstained with DAPI to detect the nuclei (blue). The intimal layer is labeled (top left corner). Bottom: Box and whisker plots depicting the ratio of phosphorylated-eNOS/total eNOS levels in the intima (P = .42), media (P < .0001), and adventitia (P = .32) from nondilated TAV and UAV aortas. Box and whisker plots display the median (middle bar), the maximum and minimum values (upper and lower bars, respectively), and the first and third quartile (bottom lines and top lines of box, respectively). eNOS mRNA expression values are normalized to the mean value of 3 internal control genes (EIF2B1, ELF1, and HMBS), and then to the mean expression value in TAV nondilated aortas. eNOS protein expression values are normalized to beta-actin protein expression. Statistical comparisons were performed with the Student t test (for normal distributions) and the Mann–Whitney U test (for nonparametric distributions). ∗P < .05; TAV: n = 25, UAV: n = 9. mRNA, Messenger RNA; TAV, tricuspid aortic valve; UAV, unicuspid aortic valve.

Figure 2

Phosphorylated endothelial nitric oxide synthase (eNOS) protein levels are increased in the intima of dilated versus normal aortas from patients with normal (ie, tricuspid) aortic valves. A, Box and whisker plot depicting the mRNA expression of NOS3 (eNOS) in normal (ND; nondilated) and dilated (D) ascending aortas from individuals with a tricuspid aortic valve (TAV) ; P = .69). B, Box and whisker plot and representative Western blot images depicting the protein concentration of eNOS in TAV-ND and TAV-D ascending aortas (P = .72). C, Box and whisker plots depicting total eNOS protein levels in the intima (P = .12), media (P = .33), and adventitia (P = .71) from normal (ND; nondilated) and dilated (D) TAV aortas. D, Top: Fluorescent images of ascending aortic tissue stained for phosphorylated-eNOS (red) and counterstained with DAPI to detect the nuclei (blue). The intimal layer is labeled (top left corner). Bottom: Box and whisker plots depicting the ratio of phosphorylated-eNOS/total eNOS levels in the intima (P = .0007), media (P = .82), and adventitia (P = .12) from normal and dilated TAV aortas. Box and whisker plots display the median (middle bar), the maximum and minimum values (upper and lower bars, respectively), and the first and third quartile (bottom lines and top lines of box, respectively). eNOS mRNA expression values are normalized to the mean value of 3 internal control genes (EIF2B1, ELF1, and HMBS), and then to the mean expression value in TAV nondilated aortas. eNOS protein expression values are normalized to beta-actin protein expression. Statistical comparisons were performed with the Student t test (for normal distributions) and the Mann–Whitney U test (for nonparametric distributions). ∗P < .05; TAV-ND: n = 25, TAV-D: n = 17. mRNA, Messenger RNA.

Figure 3

Total and phosphorylated eNOS are increased with dilatation in the ascending aorta of individuals with a unicuspid aortic valve. A, Box and whisker plot depicting the mRNA expression of NOS3 (eNOS) in normal (ND; nondilated) and dilated (D) ascending aortas from individuals with a unicuspid aortic valve (UAV; P = .80). B, Box and whisker plot and representative Western blot images depicting the protein concentration of eNOS in UAV-ND and TAV-D ascending aortas (P = .04). C, Box and whisker plots depicting total eNOS protein levels in the intima (P = .002), media (P = .004), and adventitia (P = .32) from normal and dilated UAV aortas. D, Top: Fluorescent images of ascending aortic tissue stained for phosphorylated-eNOS (red) and counterstained with DAPI to detect the nuclei (blue). The intimal layer is labeled (top left corner). Bottom: Box and whisker plots depicting the ratio of phosphorylated-eNOS/total eNOS levels in the intima (P = .0006), media (P = .03), and adventitia (P = .02) from normal and dilated UAV aortas. Box and whisker plots display the median (middle bar), the maximum and minimum values (upper and lower bars, respectively), and the first and third quartile (bottom lines and top lines of box, respectively). eNOS mRNA expression values are normalized to the mean value of 3 internal control genes (EIF2B1, ELF1, and HMBS), and then to the mean expression value in TAV nondilated aortas. eNOS protein expression values are normalized to beta-actin protein expression. Statistical comparisons were performed by the Student t test (for normal distributions) and the Mann–Whitney U test (for nonparametric distributions). ∗P < .05; UAV: normal: n = 9, dilated: n = 13. eNOS, Endothelial nitric oxide synthase; mRNA, messenger RNA; UAV, unicuspid aortic valve.

Figure 4

Endothelial nitric oxide synthase (eNOS) expression is decreased in the convexity of the aortic wall in tricuspid aortic valve (TAV)-associated aortas. A, Box and whisker plots depicting the mRNA expression of NOS3 (eNOS; left; P = .73) and the protein concentration of eNOS (right; P = .71) in the convexity and the concavity of TAV-associated nondilated aortas at the level of the sinotubular junction (STJ). B-C, Box and whisker plots depicting the mRNA expression (left; P = .004) and the protein concentration of eNOS (right; P = .75) in the convexity and the concavity of TAV associated dilated aortas (B) at the level of the STJ, and (C) at the level of the mid-ascending aorta (Mid-Asc. Ao.); mRNA: left; P = .004, protein: right; P = .67). Box and whisker plots display the median (middle bar), the maximum and minimum values (upper and lower bars, respectively), and the first and third quartile (bottom lines and top lines of box, respectively). eNOS mRNA expression values are normalized to the mean value of 3 internal control genes (EIF2B1, ELF1, and HMBS). eNOS protein expression values are normalized to beta-actin protein expression. Statistical comparisons were performed with the Student t test (for normal distributions) and the Mann–Whitney U test (for nonparametric distributions). TAV: nondilated: n = 25, dilated: n = 17. ∗P < .05. mRNA, Messenger RNA.

Figure 5

Endothelial nitric oxide synthase (eNOS) is similar in the convex and concave aortic walls in unicuspid aortic valve (UAV)-associated aortas. A, Box and whisker plots depicting the mRNA expression of NOS3 (eNOS; left; P = .12) and the protein concentration of eNOS (right; P = .13) in the convexity and the concavity of unicuspid aortic valve (UAV)-associated nondilated aortas at the level of the sinotubular junction (STJ). B-C, Box and whisker plots depicting the mRNA expression (left; P = .89) and the protein concentration of eNOS (right; P = .87) in the convexity and the concavity of UAV associated dilated aortas (B) at the level of the sinotubular junction, and (C) at the level of the mid-ascending aorta (Mid-Asc. Ao.); mRNA: left; P = .10, protein: right; P = .14). Box and whisker plots display the median (middle bar), the maximum and minimum values (upper and lower bars, respectively), and the first and third quartile (bottom lines and top lines of box, respectively). eNOS mRNA expression values are normalized to the mean value of 3 internal control genes (EIF2B1, ELF1, and HMBS). eNOS protein expression values are normalized to beta-actin protein expression. Statistical comparisons were performed by the Student t test (for normal distributions) and the Mann–Whitney U test (for nonparametric distributions). Data are presented as mean (bars) ± standard error of the mean (error bars). UAV: nondilated: n = 9, dilated: n = 13. ∗P < .05. mRNA, Messenger RNA.

Figure 6

The expression patterns of endothelial nitric oxide synthase (eNOS) and phosphorylated eNOS in the normal and dilated ascending aorta associated with tricuspid and unicuspid aortic valves. Tissue samples were taken from the convex, concave and anterior walls of normal or dilated ascending aortas from individuals with a tricuspid aortic valve (TAV) or with a unicuspid aortic valve (UAV). At the anterior circumference, the total eNOS (endothelial nitric oxide synthase) mRNA and protein were measured and compared between groups. eNOS mRNA was unchanged (=) between groups, whereas eNOS protein was decreased (↓) in the intimal layer of normal-sized UAV aortas. eNOS protein increased (↑) in the intimal and medial layers with dilatation in the UAV aortas. Phosphorylated-eNOS (p-eNOS) was also measured at the anterior circumference. In dilated TAV aortas, p-eNOS was increased in the intima (Int). In UAV aortas, p-eNOS was increased in the medial layer, and it was increased in the intima, media (Med) and adventitia (Adv) of dilated UAV aortas. Total eNOS mRNA and protein and p-eNOS protein were compared at the convexity and the concavity within each aorta. In dilated TAV aortas, eNOS mRNA was increased at the concavity versus the convexity. No other changes were observed when comparing the convex and concave walls of the aorta for either group. =: no statistical difference between groups (P ≥ .05). ↑: increased value with statistical significance (P < .05). ↓: decreased value with statistical significance (P < .05). mRNA, Messenger RNA.

Figure E1

Comparison of original analysis and subanalysis after correcting for age. A, Graph depicting patient age for all patients with a tricuspid aortic valve (TAV) and with a unicuspid aortic valve (UAV). ∗P < .0001. B, Box and whisker plots comparing the mRNA (left) and protein (right) concentrations between groups with the original data set. ∗P = .02 and .04. TAV-ND: TAV–nondilated: N = 25, TAV-D: TAV–dilated: N = 17, UAV-ND: UAV–nondilated: N = 9, UAV-D: UAV–dilated: N = 13. C, Graph depicting patient age of TAV and UAV patients after removing the oldest 10 patients from the TAV group and the youngest 5 patients from the UAV group. P = .08. D, Box and whisker plots comparing the mRNA (left) and protein (right) concentrations between groups with the age-adjusted data set. ∗P = .003 and .011. TAV-ND: TAV–nondilated: N = 17, TAV-D: TAV–dilated: N = 14, UAV-ND: UAV–nondilated: N = 7, UAV-D: UAV–dilated: N = 10. In both the original data set and the age-adjusted data set, significant differences between groups were observed only in eNOS protein concentration between the TAV-ND and UAV-ND groups and the UAV-ND and UAV-D groups. eNOS, Endothelial nitric oxide synthase; mRNA, messenger RNA.